[go: up one dir, main page]

WO2018190357A1 - Procédé d'immobilisation de lectine - Google Patents

Procédé d'immobilisation de lectine Download PDF

Info

Publication number
WO2018190357A1
WO2018190357A1 PCT/JP2018/015163 JP2018015163W WO2018190357A1 WO 2018190357 A1 WO2018190357 A1 WO 2018190357A1 JP 2018015163 W JP2018015163 W JP 2018015163W WO 2018190357 A1 WO2018190357 A1 WO 2018190357A1
Authority
WO
WIPO (PCT)
Prior art keywords
lectin
peptide
tagged
sugar chain
tag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2018/015163
Other languages
English (en)
Japanese (ja)
Inventor
浩章 舘野
淳子 片山
一隆 的場
陽一 熊田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyoto Institute of Technology NUC
Nissan Chemical Corp
National Institute of Advanced Industrial Science and Technology AIST
Original Assignee
Kyoto Institute of Technology NUC
Nissan Chemical Corp
National Institute of Advanced Industrial Science and Technology AIST
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyoto Institute of Technology NUC, Nissan Chemical Corp, National Institute of Advanced Industrial Science and Technology AIST filed Critical Kyoto Institute of Technology NUC
Priority to JP2019512540A priority Critical patent/JP7228163B2/ja
Priority to US16/604,565 priority patent/US11078254B2/en
Publication of WO2018190357A1 publication Critical patent/WO2018190357A1/fr
Anticipated expiration legal-status Critical
Priority to JP2022172853A priority patent/JP2023011764A/ja
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

  • the present invention relates to a substrate on which a lectin is immobilized, a method for immobilizing a lectin on a substrate, a target sugar chain using the substrate, a target glycoconjugate or a cell having a target complex carbohydrate, extracellular
  • the present invention relates to a method for detecting, measuring or separating vesicles or viruses (hereinafter collectively referred to as “target sugar chain-containing antigen”).
  • sugar chains The surface of living cells is covered with sugar chains, and it is thought that information is exchanged with other cells via sugar chains. Changes in response to changes in the state of cells in various situations such as pathogen infection and canceration. Therefore, by analyzing the sugar chains on the cell surface, it is possible to know the degree of cell differentiation, the presence or absence of cancer or pathogens, and the severity of the disease. Also, in the development of drugs for cancer and various diseases, analysis of sugar chain antigens is important as a therapeutic target or for elucidating the cause of side effects.
  • WFA-binding glycoprotein intrahepatic cholangiocarcinoma surface marker Patent No. 5787389
  • WFA WFA-binding glycoprotein intrahepatic cholangiocarcinoma surface marker
  • BPL BPL Examples include diagnostic techniques for various diseases such as detection of a sugar chain marker for liver disease pathology comprising a lectin-binding M2BP glycoprotein such as (patent No. 5031928).
  • BC2LCN is a lectin that can specifically recognize undifferentiated sugar chain markers “Fuc ⁇ 1-2Gal ⁇ 1-3GlcNAc” and / or “Fuc ⁇ 1-2Gal ⁇ 1-3GalNAc” on the surface of stem cells such as iPS cells.
  • the degree of undifferentiation of stem cells can be determined by measuring the BC2LCN-specific sugar chain contained in podocalyxin in the culture supernatant, and culturing stem cells using the BC2LCN lectin probe.
  • a lectin-lectin sandwich method and a lectin-antibody sandwich ELISA method for determining the degree of undifferentiation in the supernatant have been developed (Patent Document 1).
  • the present inventors obtained undifferentiated cells from a stem cell culture solution after differentiation treatment such as stem cell concentration technology using BC2LCN-immobilized carrier and biological transplantation materials.
  • a technology for separation and removal has also been developed (Patent Document 2).
  • the interaction between lectins and sugar chains is extremely low, and is 100 to 10,000 times smaller than the binding constant of antigen-antibody reaction (generally, the binding constant of antigen-antibody reaction is 10 6-9 M -1 whereas the binding constant between the lectin and the sugar chain is 10 4-7 M -1 ). Therefore, there are many cases where sufficient washing cannot be performed on the lectin bound to the target sugar chain antigen, and it has been difficult to perform accurate measurement because of high background in general fluorescence detection equipment such as ELISA and immunoassay detection equipment. .
  • the present invention provides a lectin plate that can be sufficiently washed after binding to a target sugar chain-containing antigen, as well as an antibody plate, has a stable quality, is highly sensitive, and is inexpensive, and for producing the lectin plate.
  • An object of the present invention is to provide a method for immobilizing lectins.
  • Patent Documents 3 to 6 A group of peptides having specific adsorption ability was developed (Patent Documents 3 to 6). Particularly in the case of low molecular weight antibodies such as single-chain antibodies, high affinity specific to the target antigen can be exerted by immobilizing the substrate surface with a high density and high orientation by this method ( Non-patent document 1, Patent document 7).
  • the peptide group mainly having affinity for hydrophilic polystyrene resin described in Patent Document 3 is referred to as “PS tag”, and the polycarbonate and / or polymethyl methacrylate resin affinity peptide group described in Patent Document 4 is used.
  • PS tag polycarbonate and / or polymethyl methacrylate resin affinity peptide group described in Patent Document 4
  • PMMA / PC tag silicon nitride (Si 3 N 4 ) affinity peptide group described in Patent Document 5 as “SiN tag”
  • PDMS polydimethylsiloxane
  • Tag is described. That is, the term “PS tag” in the present invention may refer to all “lectin immobilization tags” of the present invention including “PMMA / PC tags”, “SiN tags” and “PDMS tags”.
  • a lectin is a protein having a bulky and complicated steric structure and a variety of charge states on the surface, so it is unclear how densely it can be immobilized. In addition, it was unclear whether densification was effective in the first place. Furthermore, since lectins usually have multiple target recognition sites due to multimerization, it was also unclear whether the affinity could be improved by orientation control.
  • BC2LCN SEQ ID NO: 24
  • a PS tag a PSI tag (RIIIRRIRR: SEQ ID NO: 2) and a PSS tag (RSSSRRSRR: SEQ ID NO: 9) are used, and a PSI tag or PSS is used on the N-terminal side or C-terminal side of BC2LCN lectin using gene recombination technology.
  • RIIIRRIRR SEQ ID NO: 2
  • SSRRSRR PSSSRRSRR: SEQ ID NO: 9
  • PS tag has a PS tag corresponding to the type of lectin. It was found that there is a compatibility. That is, it was found that a PS tag having a good compatibility could be selected as appropriate depending on the type of lectin to be immobilized.
  • a BC2LCN lectin plate with high density orientation was prepared by adsorbing the C2 end of BC2LCN lectin to a polystyrene plate modified with PSS tag or PSI tag and modified with high hydrophilicity. Can withstand sufficient cleaning.
  • PSS / PSI-tagged lectin plates have lot-to-lot differences compared to the plates used for immobilization in biotin-avidin system, in addition to the advantage of eliminating the need for avidin coating in advance and cost. There is a remarkable effect that a uniform plate can be produced.
  • the obtained PSS / PSI-tagged BC2LCN lectin plate was applied to the ELISA method to detect undifferentiated cell glycan markers in the iPS cell culture supernatant and compared with the detection result by the conventional biotin-avidin binding plate
  • high reactivity detection sensitivity
  • the detection limit value can be significantly lowered and the variation in data can be reduced by selecting the blocking agent and optimizing the blocking conditions.
  • a high reactivity and a low detection limit value could be achieved.
  • a fusion of various lectins and a tag peptide such as “PS tag” is also simply referred to as “lectin-peptide fusion” or “tagged lectin”.
  • the present invention includes the following inventions.
  • [1] A lectin-peptide fusion having a peptide having an adsorption function on the substrate surface on the N-terminal side or C-terminal side of a lectin that recognizes a target sugar chain.
  • [2] The fusion according to [1], wherein the peptide is a peptide selected from the group consisting of any one of PS peptide, PMMA / PC peptide, SiN peptide, and PDMS peptide.
  • the lectin is rACG, rPSL1a, rLSLN, rDiscoidin I, rDiscoidin II, rCGL2, rSRL, rF17AG, rGRFT, rOrysata, rCalsepa, rBC2LA, rAAL, rPAIIL, rRSIIL, rPPL, rCNL, rPAR, ARL
  • a lectin-peptide fusion gene comprising a base sequence encoding the amino acid sequence according to [6].
  • a vector comprising the lectin-peptide fusion gene according to [7] above and capable of expressing a lectin-peptide fusion.
  • An immobilized lectin-peptide fusion wherein the peptide side in the lectin-peptide fusion according to any one of [1] to [6] is immobilized on a substrate.
  • a resin substrate having a hydrophilic surface is preferable as the substrate.
  • a substrate comprising a resin plate having a hydrophilic surface on which the lectin-PS tag fusion according to [5] or [6] is immobilized.
  • a blocking agent is contained in the diluted solution of the substrate on which the lectin-peptide fusion is immobilized and / or in the diluted antibody solution.
  • a step of adsorbing a sample containing a cell having a target sugar chain on the cell surface or a complex carbohydrate having a target sugar chain to the substrate according to [10], and a step of recovering a target sugar chain-containing material A method for concentrating cells or complex carbohydrates having a target sugar chain, comprising: [16] A kit or apparatus for measuring or isolating a target sugar chain-containing antigen comprising the substrate according to [10]. [17] The kit or device according to [16], further comprising an antibody that recognizes the target sugar chain-containing antigen.
  • a method for producing a substrate on which a lectin-peptide fusion is immobilized comprising a step of bringing the lectin-peptide fusion according to any one of [1] to [6] into contact with the substrate.
  • the production method according to [18], wherein the contacting step is performed in a buffer solution.
  • a highly sensitive lectin plate with high density and orientation can be provided at low cost, and a highly accurate lectin-lectin sandwich assay and lectin-antibody sandwich assay can be easily performed. Since complex carbohydrates containing target sugar chain markers can be detected and measured simply, quickly and accurately, cancer and various diseases can be easily and accurately diagnosed at medical sites.
  • the high-density, highly aligned lectin substrate of the present invention glycoproteins and cells containing the target sugar chain can be separated more quickly and accurately. For example, by using it for the isolation process of stem cells or various somatic cells differentiated from stem cells, it will lead to the provision of safe transplant materials derived from stem cells in future regenerative medicine.
  • micro BCA After reacting with various plates when suspended in PBS or PBST as in the case of rBC2LCN lectin for 1 hour at room temperature and washing 5 times with PBST The adsorption amount by assay (ThemoFisher) was measured. Since the effect of the highly hydrophilic carbo plate and AGC plate was the same as that of the rBC2LCN lectin, the figure shows only the adsorption amount of the carbo plate and the AGC plate. The same applies to the following lectins.
  • Amounts adsorbed at various concentrations on carboplates and AGC plates when untagged, PSI-tagged, or PSS-tagged rGal3C lectin is suspended in PBS or PBST.
  • Adsorption amounts at various concentrations on carboplates and AGC plates when untagged, PSI-tagged, and PSS-tagged rPSL1a lectins are suspended in PBS or PBST.
  • Adsorption amounts at various concentrations on carboplates and AGC plates when untagged, PSI-tagged, and PSS-tagged rPALa lectins are suspended in PBS or PBST.
  • Lectin immobilization tag used in the present invention (1-1) Types of lectin immobilization tag used in the present invention
  • the lectin immobilization tag used in the present invention was previously developed by the present inventors.
  • “PS tag” consisting of hydrophilic polystyrene affinity peptide group (Patent Document 3), “PMMA / PC tag” consisting of polycarbonate and / or polymethyl methacrylate resin affinity peptide group (Patent Document 4), silicon nitride (Si 3 N 4 )
  • SiN tag (patent document 5) composed of affinity peptide group and “PDMS tag” (patent document 6) composed of polydimethylsiloxane affinity peptide group are all included.
  • the substrate for immobilization In accordance with the substrate for immobilization and considering compatibility with the lectin to be used, it can be appropriately selected from these various peptide groups.
  • the method for binding to the lectin gene in the production of the tagged lectin fusion protein of the present invention and the method for expressing the tagged lectin from the host cell using the expression vector are the same, and the method for adsorption to the solid phase is also used.
  • “PS tag” is mainly described as an immobilization tag of the present invention, but is not limited to “PS tag” because it is almost common.
  • the amino acid sequence of the lectin immobilization tag used in the present invention is 10% in the amino acid sequence specifically represented by the following SEQ ID NOs within a range that does not impair the solid-phase (base material) adsorption ability. Less than%, preferably less than 8%, more preferably less than 5%, allow deletion, substitution, addition or insertion of amino acid residues.
  • Less than%, preferably less than 8%, more preferably less than 5% allow deletion, substitution, addition or insertion of amino acid residues.
  • the deletion, substitution, addition or insertion site at that time is only the C-terminal or N-terminal of the tag.
  • PS tag that can be used in the present invention is a peptide group described in Patent Document 3 that exhibits a specific adsorption function on a solid phase surface having a hydrophilic resin surface. It refers to the peptide contained, and is particularly suitable when a polystyrene resin or polycarbonate resin having an improved surface hydrophilicity is used as the solid phase. It can be applied to general polystyrene resins or polycarbonate resins in general as a solid phase for sandwich assays with antibodies such as lectin arrays, ELISA, and immunoassay methods.
  • Peptide groups represented as peptides for example, “RIIIRRIRR (SEQ ID NO: 2)” “RAIARRIRR (SEQ ID NO: 3)”, “RLLLRRLRR (SEQ ID NO: 4)”, “RVVVRRVRR (SEQ ID NO: 5)”, “RAAARRARR (sequence) No. 6), “RGGGRRGRR (SEQ ID NO: 7)”, “RMMMRRMRR (SEQ ID NO: 8)”, “RSSSRRSRR (SEQ ID NO: 9)”, or “RTTTRRTRR (SEQ ID NO: 10)”.
  • KGLRGWREMISL (sequence number 11)
  • ADYLSRWGSIRN (sequence number 12)”
  • SRVHRAVLNGVS (sequence number 13)
  • RPPGVVRRYALG (sequence number 14)”
  • VRSWEEQARVTT (sequence number 15)”
  • RAFIASRRIKRP (sequence) No. 16)
  • RESTLKGTSRAV SEQ ID NO: 17)
  • AGLRLKKAAIHR SEQ ID NO: 18
  • SSLLRAVPEPTG (SEQ ID NO: 19)
  • RAFIASRRIRRP (SEQ ID NO: 20)
  • hydrophilic resins Since it has a specific adsorption ability for a solid phase having a surface, it can be similarly used as the “PS tag” of the present invention.
  • a preferable PS tag for producing the lectin plate of the present invention is a peptide containing “RIIIRRIRR (SEQ ID NO: 2)” or “RSSSRRSRR (SEQ ID NO: 9)”, the former being a PSI tag and the latter being a PSS. Sometimes called a tag.
  • PS tags have compatibility with each type of lectin, it is preferable to select and use a PS tag with good compatibility as appropriate according to the type of lectin to be immobilized.
  • PMMA / PC tag that can be used in the present invention has a high solid phase affinity mainly in the case of a solid phase whose solid surface is a polycarbonate or polymethyl methacrylate resin.
  • Polycarbonate is widely used as an engineering plastic, and polymethyl methacrylate resin is used as a substrate such as a protein chip (Patent Document 4).
  • Patent Document 4 a peptide tag including “SEQ ID NO: 21 (DVEGIGDVDLVNYFEVGATYYFNK)”, which is referred to as PMOMP25 peptide in Patent Document 4, can be mentioned, but the invention is not limited thereto. Any of the peptides shown in can be used.
  • SiN tag The PC tag that can be used in the present invention has high solid-phase affinity when the solid-phase surface is silicon nitride (Si 3 N 4 ). Silicon nitride is widely used as a semiconductor material (Patent Document 5). Specifically, among SiN tags, a peptide tag including “SEQ ID NO: 22 (GGRHTPFFKGYRPQFYFRTTDVTGTIELPE)” referred to as V821 peptide in Patent Document 5 can be mentioned, but the present invention is not limited to this and is shown in Patent Document 5. Any peptide can be used.
  • the PDMS tag that can be used in the present invention has a high solid phase affinity when the solid phase surface is a solid phase that is polydimethylsiloxane, which is a kind of silicone rubber.
  • Polydimethylsiloxane is known as one of microchip substrates such as microchannels (Patent Document 6).
  • a peptide tag including “SEQ ID NO: 23 (MVMPGDNIKMVVTLIHPIAMDDGLRFAIRE)”, which is referred to as ELN-V81 peptide in Patent Document 6, can be mentioned. Any of the peptides shown can be used.
  • the lectin according to the present invention may be any lectin that recognizes a specific target sugar chain, but is bound to the immobilization tag of the present invention such as a PS tag.
  • a PS tag For this, it is preferable to connect a gene encoding a PS tag and the like to a lectin gene and obtain it in a recombinant form. Therefore, it is preferable that the full-length amino acid sequence or an amino acid sequence including at least a sugar chain recognition site is a known lectin.
  • Examples include rAAL2, rBambL, and rPVL lectins, but are not limited to these lectins.
  • the immobilization tag of the present invention is appropriately selected from the immobilization tags of the present invention depending on the type of lectin to be immobilized or the type of solid phase surface to be immobilized. It is preferable to select and use.
  • a gene encoding the immobilization tag of the present invention such as a PS tag is bound to the 5 ′ end or 3 ′ end of the lectin gene, the opposite side is used to facilitate the detection and purification of the recombinant tagged tag lectin.
  • it may be expressed as a fusion protein with a tag peptide such as a FLAG tag, 3 ⁇ FLAG tag, His tag (for example, 6 ⁇ His tag) or other protein at the 3 ′ end or 5 ′ end.
  • the tag peptide for immobilization of the present invention can be directly bound to the target lectin.
  • the amino acid sequence of the lectin used in the present invention does not have to be a full length as long as it retains a specific target sugar chain recognition site, and may be a partial sequence having a target sugar chain recognition site. Further, within the range that does not impair the recognition ability of a specific target sugar chain, the amino acid sequence is less than 10%, preferably less than 8%, more preferably less than 5%, still more preferably less than 3%, more preferably less than 2%. Particularly preferably, if it is less than 1%, deletion, substitution, addition or insertion of an amino acid residue is allowed.
  • amino acid residues for example, 20 or less, such as 10 or less, such as 5 or less, such as 3 or less, such as 2 or less, for example 1 amino acid residue deletion or substitution. Addition or insertion is allowed. Deletion, substitution, addition or insertion at that time is preferably only at the C-terminal or N-terminal of the amino acid sequence of the lectin.
  • the amino acid sequence of the lectin-peptide fusion used in the present invention is also less than 10% in the amino acid sequence, preferably within a range that does not impair the ability to recognize a specific target sugar chain and the ability to adsorb a solid phase (substrate). Less than 8%, more preferably less than 5%, even more preferably less than 3%, more preferably less than 2%, particularly preferably less than 1%, allow deletion, substitution, addition or insertion of amino acid residues. . In terms of the number of amino acid residues, for example, 20 or less, such as 10 or less, such as 5 or less, such as 3 or less, such as 2 or less, for example 1 amino acid residue deletion or substitution. Addition or insertion is allowed. The deletion, substitution, addition or insertion described on the left is preferably only at the C-terminal or N-terminal of the amino acid sequence of the lectin or peptide of the fusion.
  • the expression vector is not particularly limited as long as it has a function of expressing and producing the tagging lectin for immobilization of the present invention as a fusion protein in various host cells.
  • Commercially available plasmid vectors, phage vectors and viral vectors can be used.
  • host cells Escherichia coli, Bacillus subtilis, actinomycetes, yeast, filamentous fungi, plant cells, insect cells, animal cells and the like can be used, but Escherichia coli is preferred.
  • Transformation of a host cell with a recombinant expression vector can be performed using a conventionally known method, and when a commercially available Competent Cell is used. The transformation may be performed according to the product protocol.
  • a coupling reaction between a normal protein and a peptide is performed on a lectin whose base sequence is unknown, such as a naturally-derived lectin, and an immobilizing tag peptide synthesized in advance by chemical synthesis or gene recombination.
  • the present invention is not limited to these, and it is possible to obtain a fusion protein by using amine coupling or diol coupling using SH group or maleimide group. Fusion methods other than covalent bonds such as streptavidin-biotin reaction can also be used.
  • Substrate for immobilizing the lectin of the present invention (2-1) Immobilized Lectin Substrate
  • a plate eg, microwell plate
  • a microarray substrate eg, slide for microarray
  • tubes e.g, beads
  • chromatographic carriers eg, Sepharose TM
  • membranes eg, nitrocellulose membrane, PVDF membrane, polystyrene nonwoven fabric
  • gels eg, polyacrylamide) Gel
  • plates, beads and membranes are preferably used, and plates are most preferably used from the viewpoint of easy handling.
  • the solid phase of the present invention is not limited to the plate alone.
  • the plate material When using a PS tag, the plate material preferably has a hydrophilic resin surface, such as polystyrene, polycarbonate, polypropylene, polyethylene, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), etc.
  • a hydrophilic resin surface such as polystyrene, polycarbonate, polypropylene, polyethylene, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), etc.
  • a plastic resin surface that has been modified to be hydrophilic can be used. It is particularly preferable to use hydrophilic polystyrene or polycarbonate. Many of these hydrophilic resin plates are commercially available, but can be rendered hydrophilic by performing UV + O 3 treatment or plasma oxidation treatment on the hydrophobic resin surface.
  • a solution or suspension containing the tagging lectin for immobilization of the present invention is adjusted to conditions optimized according to the methods described in Patent Documents 3 to 6 according to the type and plate of the tag, and immobilized. It is brought into contact with the surface of a substrate (solid phase) such as a plate to be converted.
  • a PSI tag is used as the PS tag, the pH is preferably in the range of pH 6.5 to pH 7.5, and particularly preferably around pH 7.0 such as pH 6.8 to 7.2.
  • PSS tag When a PSS tag is used as the PS tag, it is preferably in the range of pH 6.5 to pH 7.5, particularly preferably around pH 7.5 such as pH 7.3 to 7.7.
  • the preferred salt concentration for immobilization is 0.05 to 0.30M in terms of NaCl concentration, more preferably 0.10 to 0.20M, and the most preferred concentration is around 0.15M such as 0.13 to 0.17M in terms of NaCl concentration. It is.
  • sample to be measured of the present invention By using the lectin-peptide fusion immobilization substrate of the present invention (lectin plate or the like), the target sugar chain in the sample Can be detected and / or measured (hereinafter collectively referred to as “measurement”).
  • the sample to be measured in the present invention is a target sugar chain, a complex carbohydrate containing the target sugar chain, or a cell, extracellular vesicle or virus having the target complex carbohydrate (that is, “target sugar chain-containing antigen”). ) Is a sample that may exist.
  • the target sugar chain or complex carbohydrate examples include various cell-specific sugar chains such as undifferentiated cell markers such as the surface of cultured cells, complex carbohydrates such as glycoproteins or glycolipids, or sugar chains in the culture supernatant.
  • cell-specific sugar chains such as undifferentiated cell markers such as the surface of cultured cells
  • complex carbohydrates such as glycoproteins or glycolipids, or sugar chains in the culture supernatant.
  • disease-specific sugar chains or glycoconjugates observed in body fluids such as blood of subjects and test animals, or on organs, tissues, and cell surfaces
  • the sample to be measured in the present invention is a sample that may contain these target sugar chains or target complex carbohydrates, and includes suspensions, cell lysates, cell cultures containing cultured cells or living cells themselves.
  • a supernatant or a suspension containing a body fluid such as blood derived from a subject or a test animal, or a purified solution or a diluted solution.
  • a typical measurement method to which the tagged lectin immobilization substrate of the present invention can be applied is typical.
  • a plurality of lectins can be immobilized on the same substrate and used as a lectin array. In the following, typical sandwich ELISAs are described, but not limited thereto.
  • the tagged lectin-immobilized plate of the present invention (hereinafter also simply referred to as a lectin plate) can be used as a solid phase of a sandwich ELISA.
  • the solid phase (plate) to which the PS-tagged lectin is bound is washed several times, and further washed several times after blocking.
  • the sample containing the target substance is reacted with the lectin on the solid phase
  • the sample is washed multiple times, and the overlaid antibody and the target substance are reacted to wash the sample multiple times. There are multiple washing steps after the reaction with the enzyme-labeled antibody that reacts with the overlay antibody.
  • the enzyme-labeled antibody is reacted with the substrate, the absorbance is measured, the target substance in the sample is detected, or the concentration is measured.
  • the amount of lectin is hardly reduced even when such washing steps are performed.
  • the overlaying antibody is preferably an antibody having binding activity with the protein portion of the glycoprotein containing the target sugar chain, and the antibody can also be directly labeled.
  • the labeling substance fluorescent substance (FITC, rhodamine, Cy3, Cy5, etc.), radioactive materials (such as 13 C, 3 H), enzymes (alkaline phosphatase, peroxidase, etc.), glucose oxidase, etc. ⁇ - galactosidase.
  • the antibody may be labeled with biotin
  • streptavidin may be labeled with the labeling substance
  • the binding between biotin and streptavidin may be used.
  • detection is performed using an appropriate substrate corresponding to the enzyme used.
  • o-phenylenediamine OPD
  • TMB tetramethylbenzidine
  • the measurement of the label signal may be performed using an appropriate measuring device according to the labeling substance used.
  • the lectin plate is treated with a surfactant (for example, 0.1% Tween20) before and after reacting with the labeled antibody for detection. It is preferable to wash with a PBS buffer solution. In particular, it is preferable to subject the lectin plate before the reaction to a blocking treatment with a blocking agent (for example, a PBS buffer containing 2% BSA 0.1% Tween 20). It is also effective to use the blocking agent (for example, a PBS buffer containing 2% BSA 0.1% Tween 20) as a diluted solution of the labeled antibody for detection to be reacted.
  • a surfactant for example, 0.1% Tween20
  • a PBS buffer solution for example, a PBS buffer containing 2% BSA 0.1% Tween 20
  • a blocking agent for example, a PBS buffer containing 2% BSA 0.1% Tween 20
  • a blocking agent As a blocking agent in that case, instead of the 2% BSA-containing blocking agent, a blocking agent “prevelex TM LS1004 (manufactured by Nissan Chemical Industries, Ltd.)” containing the coating film forming composition described in WO2014 / 196650 is used. However, even if Blocking One (manufactured by Nacalai Tesque), Block Master CE210, CE510 (manufactured by JSR Life Sciences) is used, the same or higher LLOD value lowering effect is exhibited.
  • ELISA kits for measuring a target sugar chain or a target complex carbohydrate are used to measure a solid phase (lectin plate) on which one or more of the PS-tagged lectins of the present invention are immobilized. It can be set as a measurement kit. Furthermore, it is preferable to combine with one or more of labeled antibodies for detection, blocking agents, buffers, diluents and the like. Instructions etc. can be added to the kit.
  • the blocking agent a blocking agent “prevelex TM LS1004 (manufactured by Nissan Chemical Industries, Ltd.)” containing the coating film forming composition described in WO2014 / 196650 is preferable. Further, it is more preferable to combine a blocking agent, particularly an antibody diluent containing a coating film-forming composition-containing blocking agent, as a diluent for the labeled antibody for detection.
  • Separation method using the tagged lectin of the present invention (4-1) Separation and recovery of cells
  • a sugar chain present on the surface of the cell to be separated and recovered is targeted. From the sample containing the cells, only the cells can be concentrated, separated and / or recovered (hereinafter also referred to as “separation”).
  • the target sugar chain-specific lectin is immobilized on a substrate (solid phase) such as a bead, particle, or plate such as a magnetic bead via the lectin immobilization tag of the present invention, and the target sugar chain is immobilized.
  • a stem cell can be isolated from a body fluid sample such as blood of a mammal such as a human or a cell-containing sample derived from a tissue by targeting a stem cell sugar chain marker or an undifferentiated cell sugar chain marker.
  • a cancer cell sugar chain marker or a sugar chain marker that is specifically expressed in a specific disease cancer cells and lesion cells can also be isolated.
  • a PMMA tag for example, SEQ ID NO: 21
  • glycoconjugates such as glycoproteins (concentration, separation, purification, recovery) Separation (concentration or isolation) can be performed in a state in which it is bound to lectin by a method similar to that for measuring glycoconjugates such as glycoproteins.
  • the recovery of glycoconjugates from lectins can be easily recovered by allowing each lectin-specific monosaccharide solution to act.
  • a solid phase such as a magnetic bead such as a magnetic bead, a column or a plate on which one or more kinds of lectins with a PS tag (preferably PMMA tag) of the present invention are immobilized is used as a target sugar chain or target sugar.
  • a kit for concentrating, separating, and purifying a chain-containing material (a target glycoconjugate, a cell having a target glycoconjugate, an extracellular vesicle, a virus, or the like) can be obtained.
  • kits in combination with one or more kinds of other lectins or antibodies that recognize the target sugar chain or target glycoconjugate, a blocking agent, a buffer solution, a diluent, and the like. Instructions for use can be added to the kit.
  • a blocking agent particularly a coating film-forming composition-containing blocking agent.
  • instruments, devices, and the like including a separation column and a separation filter can be included in the kit.
  • Example 1 Synthesis of PSS-tagged rBC2LCN and PSI-tagged rBC2LCN lectin
  • rBC2LCN lectin was used as a lectin, and a PSS tag (SEQ ID NO: 9) together with a typical PSI tag (SEQ ID NO: 2) as a PS tag. ) was selected, and four types of constructs bound to the N-terminal side or C-terminal side of the rBC2LCN lectin were synthesized.
  • a PSI tag SEQ ID NO: 2 or 5 ′ end side or 3 ′ end side of a gene encoding BC2LCN (SEQ ID NO: 24; GenBank / NCBI-GI registration number: YP_002232818 (Genome ID: 206562055)) or Four types of constructs (Fig. 1) were synthesized by linking a gene encoding one of the PSS tags (SEQ ID NO: 9), and FLAG tags bound to the opposite ends of each, and each construct was constructed into a pET27b vector. Inserted. Subsequently, it was introduced into an E. coli (BL21-CodonPlus) host, and BC2LCN added with four corresponding PS tags was expressed.
  • E. coli BL21-CodonPlus
  • Example 2 Yield comparison of recombinant PS-tagged lectins As lectins, rACG, rPSL1a, rLSLN, rDiscoidin I, rDiscoidin II, rCGL2, rSRL, rF17AG, rGRFT, rOrysata, rCalsepa, rBC2LA, rAAL, rPAIIr, RS Select rCNL, rPAIL, rABA, rMOA, rPALa, rGal3CS, rMpL, rAAL2, rBambL, rPVL, so that the PSI tag or PSS tag is bound to the C-terminal side, and the FLAG tag is bound to the opposite side.
  • Example 3 Adsorption amount of PS-tagged lectin to various plates (3-1) rBC2LCN lectin rBC2LCN lectin that is not tagged, PSI-tagged, or PSS-tagged, PBS or PBS containing 0.1% Tween20 (PBST) And reacted with various plates at room temperature for 1 hour at different concentrations.
  • a hydrazide group was contained by surface treatment of Nunc (260860) (manufactured by Thermo), Polysorp (manufactured by Thermo) and Maxisorp (manufactured by Thermo) and polystyrene plate for ELISA.
  • Carboplate manufactured by Sumitomo Bakelite Co., Ltd.
  • an AGC plate made by glass for tissue culture, manufactured by Iwaki Co., Ltd.
  • the hydrophilicity is AGC, carbo> Maxisorp, Nunc (260860) >> Polysorp.
  • the amount of lectin adsorbed on the surface of each plate was measured using a protein quantification kit (ThemoFisher) of micro BCA assay (FIG. 5).
  • rDiscoidinII lectin For untagged, PSI-tagged, or PSS-tagged rDiscoidinII lectin, the same method as in (3-1) was applied to measure the amount of lectin adsorbed on each plate surface did. As a result, rDiscoidinII lectin also showed the same tendency as rACG lectin in (3-2) (FIG. 8).
  • rPALa lectin For untagged, PSI-tagged, or PSS-tagged rPALa lectin, the amount of lectin adsorbed on the surface of each plate is measured by applying the same method as in (3-1) did. As a result, in the case of rPALa lectin, in the presence of Tween20 (PBST), a high degree of adsorption is observed when the concentration is high when PS is tagged, and almost no adsorption is observed when Ps is not tagged (FIG. 18). ).
  • rPA1L lectin For untagged, PSI-tagged, or PSS-tagged rPA1L lectin, the amount of lectin adsorbed on each plate surface is measured by applying the same method as in (3-1) did. As a result, in the case of rPA1L lectin, in the presence of Tween20 (PBST), it showed adsorptivity when PS-tagged, but hardly adsorbed without PS tag. Among PS tags, the adsorptivity in the case of PSI tags was high (FIG. 20).
  • Example 4 Optimization conditions for immobilizing lectins (4-1) pH optimization conditions Detection of undifferentiated sugar chain markers in culture supernatant using PSS-tagged rBC2LCN lectin and PSI-tagged rBC2LCN lectin
  • pH of the buffer solution when immobilizing the lectin was optimized.
  • PSS-tagged rBC2LCN lectin or PSI-tagged rBC2LCN lectin was adjusted to a concentration of 0.5 ⁇ g / mL in various pH buffer solutions containing 0.1% Tween20 and 0.15 M NaCl, and the carboplate for ELISA (Sumitomo Bakelite) 50 ⁇ L, and allowed to stand at room temperature for 1 hour.
  • the preferable salt concentration at the time of immobilization is preferably 0.10 to 0.20 M in terms of NaCl concentration, and particularly preferably adjusted to around 0.15 M.
  • Example 5 Comparison of LLOD value and variation degree between PS tagging method and biotinylation method
  • PSS or PSI tagged lectin plate by PS tagging method and avidin plate to which biotinylated lectin was bound were used.
  • the lower limit of detection (LLOD) and the degree of variation (CV) are compared with the undifferentiated sugar chain marker in the culture supernatant of human iPS cell strain 201B7.
  • test sample (iPS cells) was diluted with a medium (mTeSR1), applied with 50 ⁇ L / well, incubated at room temperature for 1 hour, and washed with a buffer solution. Subsequently, an HRP-labeled antibody (R10G (NH2)) was adjusted with a buffer (2% BSA / 0.1% Tween20 PBS) and applied at 50 ⁇ l / well. The plate was incubated at room temperature for 1 hour, washed with buffer, TMB solution was applied at 50 ⁇ l / well and incubated at room temperature.
  • mTeSR1 medium
  • HRP-labeled antibody R10G (NH2)
  • the reaction was stopped with 50 ⁇ l / well of 1N HCL, and the absorbance was measured by scanning with a plate reader (main wavelength 450 nm, subwavelength 620 nm). The above operation was performed in triplicate and the same operation was repeated three times.
  • Biotinylated rBC2LCN was diluted to 0.3 ⁇ g / mL with PBS, applied at 50 ⁇ L / well, incubated for 1 hour at room temperature, and then the plate was washed with a buffer.
  • a test sample (iPS cells) was diluted with a medium (mTeSR1), applied with 50 ⁇ L / well, incubated at room temperature for 1 hour, and washed 5 times with a buffer solution. Subsequently, 1 ⁇ g / mL of HRP-labeled antibody (R10G (NH 2)) was adjusted with a buffer (2% BSA / 0.1% Tween 20 PBS), and 50 ⁇ l / well was applied.
  • the plate was incubated at room temperature for 1 hour, washed with buffer, TMB solution was applied at 50 ⁇ l / well, and incubated at room temperature for 30 minutes.
  • the reaction was stopped with 50 ⁇ l / well of 1N HCL, and the absorbance was measured by scanning with a plate reader (main wavelength 450 nm, subwavelength 620 nm). The above operation was performed in triplicate and the same operation was repeated three times.
  • Example 6 Similar to the method of comparing the reactivity of PS-tagged rBC2LCN lectin and iPS cells (Example 1), rBC2LCN having a PSI-tagged or PSS-tagged C-terminal side was prepared, and no PS tag was added. It was suspended in PBS or PBST together with rBC2LCN (0.25 ⁇ g / well) and immobilized on a carboplate (Sumitomo Bakelite) at room temperature for 1 hour. After washing with PBST, 2% BSA / PBST was added at 250 ⁇ L / well and blocked for 1 hour at room temperature.
  • the hydrophobic fraction prepared from human iPS cell 201B7 was biotinylated and reacted at different concentrations for 1 hour at room temperature. After adding 50 ⁇ L / well of 10 ng / mL peroxidase-labeled streptavidin and reacting at room temperature for 1 hour, the substrate was added to develop color. After 30 minutes at room temperature, 50 ⁇ L / well of 1N hydrochloric acid was added to stop the reaction, and measurement was performed at a main wavelength of 450 nm / subwavelength of 620 nm (FIG. 26).
  • PSS-tagged rBC2LCN shows a significantly higher signal, and PSS-tagged rBC2LCN is more reactive among them.
  • the PSS-tagged rBC2LCN showed almost the same level of stable reactivity even in the presence of Tween20 (PBST) (FIG. 27). This suggests that PSS-tagged rBC2LCN can maintain high reactivity with the target sugar chain even if there are contaminants derived from the test sample in the reaction system.
  • the target sugar chain on the cell surface is directly detected using a cell lysate
  • a technique is generally used in which the cell lysate or its hydrophobic fraction is labeled with biotin and detected with HRP-labeled streptavidin.
  • the avidin plate cannot be used as the lectin plate, the usefulness of the PSS / PSI-tagged lectin plate is extremely high in that respect.
  • PS tag-free rBC2LCN lectin used in the lower limit of detection (Example 7) PS tag-free, PSI or PSS-tagged rBC2LCN was suspended in PBS or PBST (0.25 ⁇ g / well) at room temperature for 1 hour. After immobilization on a carboplate (manufactured by Sumitomo Bakelite), human iPS cells 201B7 were reacted at different concentrations, and the lower limit of detection (LLOD) was measured. As a result, it was found that rBC2LCN tagged with PSS / PSI showed a sensitivity 10 to 200 times higher than that without tag.
  • Example 8 Comparison of reactivity between various lectins and undifferentiated sugar chain markers
  • Various PSS / PSI-tagged lectins suspended in PBS or PBST were placed on Carboplate (Sumitomo Bakelite) at 100 ⁇ g / well for 1 hour at room temperature. Immobilized. After washing with PBST, 2% BSA / PBST was added at 250 ⁇ L / well and blocked for 1 hour at room temperature. Hydrophobic fractions prepared from human iPS cells 201B7 or human skin fibroblasts (obtained from ATCC) were biotinylated and reacted at different concentrations for 1 hour at room temperature.
  • Example 9 Search of optimization conditions for minimizing LLOD value and data variation of PS-tagged lectin-immobilized plate
  • human iPS cells used in (Example 6) and PS-tagged rBC2LCN In combination with an ELISA carboplate with immobilized lectin, we examined optimization of the washing solution and blocking solution in each step of the reaction so that the LLOD value of the reaction intensity measurement and the degree of variation in data were minimized. The results were compared with those obtained using an avidin plate on which biotinylated lectins were immobilized.
  • PS-tagged lectin (rBC2LCN) is adjusted to 0.5 ⁇ g / mL with buffer solution (50 mM HEPES / 150 mM NaCl 0.1% Tween20), and 50 ⁇ L to each well of ELISA carboplate (S-BIO MS-8708F) Apply. After standing at room temperature for 1 hour, the immobilized PS-tagged lectin is washed (0.1% Tween20 / PBS 350 ⁇ L / well, hereinafter simply referred to as “0.1% Tween20 / PBS”), and the washing solution is removed.
  • buffer solution 50 mM HEPES / 150 mM NaCl 0.1% Tween20
  • S-BIO MS-8708F ELISA carboplate
  • ⁇ Condition 2> In the same manner as in ⁇ Condition 1>, PS-tagged lectin was immobilized in each well of an ELISA carboplate (S-BIO MS-8708F), washed (0.1% Tween20 / PBS), and subjected to a washing solution removal step. A step of reacting a dilution series of test cells (iPS cells: 201B7 sup) in each well, washing (0.1% Tween20 / PBS), and washing solution removal step are performed.
  • ⁇ Condition 3> In the same manner as in ⁇ Condition 1>, the PS-tagged lectin is immobilized in each well of an ELISA carboplate (S-BIO MS-8708F), followed by washing (0.1% Tween20 / PBS) and washing solution removal step. Subsequently, 250 ⁇ L of 2% BSA / 0.1% Tween20 PBS is applied to each well, left at room temperature for 1 hour, and washed with 350 ⁇ L / well of PBS to remove the washing solution.
  • S-BIO MS-8708F ELISA carboplate
  • the avidin plate (blocking-less type S-BIO BS-X7603) was washed twice with a wash solution (0.1% Tween20 / PBS) at 350 ⁇ L / well, and biotinylated rBC2LCN adjusted to 0.3 ⁇ g / mL with PBS buffer was added to each. Apply 50 ⁇ l in each well. After standing at room temperature for 1 hour, 0.1% Tween20 / PBS is used as a washing solution, and washing is performed 5 times at 350 ⁇ L / well to remove the washing solution.
  • iPS cells 201B7 sup
  • 50 ⁇ L is applied to each well and allowed to stand at room temperature for 1 hour.
  • Example 10 Search for blocking agent that minimizes LLOD value of PS-tagged lectin-immobilized plate (10-1) Examination of various blocking agents In this experiment, human iPS cells and PS were examined in the same manner as in (Example 9). Using a combination with a carboplate for ELISA to which tagged rBC2LCN lectin is immobilized, the LLOD value of the reaction intensity measurement is minimized in the same protocol as in condition 3 in which the lowest LLOD was obtained in (Example 9). A search for blocking agents was performed.
  • PS-tagged rBC2LCN lectin was immobilized on each well of an ELISA carboplate (S-BIO MS-8708F) and then blocked as shown below (Table 1).
  • a detection peroxidase-labeled antibody at 1 ⁇ g / mL using a 10-fold diluted 0.1% Tween20 blocking agent and other agents, and follow the protocol in condition 3 with a plate reader (main wavelength 450 nm, subwavelength 620 nm) The absorbance was measured with (FIG. 32).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Inorganic Chemistry (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la préparation d'un matériau de base hautement sensible et peu coûteux immobilisé sur de la lectine (par exemple, une plaque de lectine), ledit matériau de base immobilisé sur de la lectine ayant des qualités stables et étant capable d'être suffisamment lavé après qu'un antigène contenant une chaîne de sucre cible s'y lie. Un autre but de la présente invention consiste à fournir un procédé d'immobilisation de lectine à un matériau de base correspondant. La présente invention concerne un procédé moyennant quoi une fusion lectine-peptide, dans laquelle un peptide capable d'adsorption à une surface de matériau de base telle qu'une étiquette PS est fusionné avec le côté de terminaison N ou le côté de terminaison C de la lectine capable de reconnaître une chaîne de sucre cible, est immobilisée sur le côté peptide à un matériau de base ; et un matériau de base immobilisé sur de la lectine produit selon ce procédé. En utilisant le matériau de base immobilisé sur de la lectine, un antigène contenant une chaîne de sucre cible peut être mesuré de manière fortement sensible et uniforme et, en outre, des cellules contenant la chaîne de sucre cible, etc., peuvent être séparées (concentrées et récoltées).
PCT/JP2018/015163 2017-04-11 2018-04-11 Procédé d'immobilisation de lectine Ceased WO2018190357A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2019512540A JP7228163B2 (ja) 2017-04-11 2018-04-11 レクチンの固定化方法
US16/604,565 US11078254B2 (en) 2017-04-11 2018-04-11 Method for immobilizing lectin
JP2022172853A JP2023011764A (ja) 2017-04-11 2022-10-28 レクチンの固定化方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017-078414 2017-04-11
JP2017078414 2017-04-11

Publications (1)

Publication Number Publication Date
WO2018190357A1 true WO2018190357A1 (fr) 2018-10-18

Family

ID=63792487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/015163 Ceased WO2018190357A1 (fr) 2017-04-11 2018-04-11 Procédé d'immobilisation de lectine

Country Status (4)

Country Link
US (1) US11078254B2 (fr)
JP (2) JP7228163B2 (fr)
TW (1) TW201841935A (fr)
WO (1) WO2018190357A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020218427A1 (fr) * 2019-04-25 2020-10-29 日産化学株式会社 Procédé d'évaluation de la qualité de cellules souches somatiques
WO2021070934A1 (fr) 2019-10-11 2021-04-15 国立研究開発法人産業技術総合研究所 Procédé de dépistage du cancer
JP2022122816A (ja) * 2021-02-10 2022-08-23 東ソー株式会社 フコース結合性タンパク質の製造方法
WO2025169902A1 (fr) * 2024-02-05 2025-08-14 国立研究開発法人産業技術総合研究所 Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111217893A (zh) * 2020-01-13 2020-06-02 黑龙江大学 一种黑木耳凝集素及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009101807A1 (fr) * 2008-02-14 2009-08-20 National University Corporation Kyoto Institute Of Technology Peptide, utilisation du peptide, procédé de fabrication du peptide, support solide ayant le peptide immobilisé sur celui-ci et procédé de fabrication du support solide
JP2011168505A (ja) * 2010-02-16 2011-09-01 Kyoto Institute Of Technology ポリカーボネートおよび／またはポリメタクリル酸メチル親和性ペプチド、およびその利用
WO2013065302A1 (fr) * 2011-11-01 2013-05-10 独立行政法人産業技術総合研究所 Procédé de détection de cellules indifférenciées et procédé de détection d'hydrate de carbone complexe
WO2013122061A1 (fr) * 2012-02-13 2013-08-22 国立大学法人京都工芸繊維大学 Peptide ayant une affinité pour le nitrure de silicium (si3n4) et son utilisation
WO2016129695A1 (fr) * 2015-02-13 2016-08-18 国立大学法人京都工芸繊維大学 Peptides ayant une affinité pour le polydiméthylsiloxane et utilisations associées

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA06006406A (es) * 2003-12-05 2007-03-21 Adnexus Therapeutics Inc Inhibidores de receptores de factor de crecimiento endotelial vascular tipo 2.
US8883985B2 (en) 2010-02-16 2014-11-11 National University Corporation Kyoto Institute Of Technology Antibody-immobilized carrier, method of producing antibody-immobilized carrier, and use of said antibody-immobilized carrier
WO2011153464A2 (fr) * 2010-06-03 2011-12-08 New York University Immobilisation orientée in situ de protéines sur un support
US9529003B2 (en) * 2011-08-26 2016-12-27 Alan Marc Kleinfeld Development and use of fluorescent probes of unbound bilirubin
WO2013128914A1 (fr) 2012-02-28 2013-09-06 独立行政法人産業技術総合研究所 Procédé d'essai de différenciation cellulaire, procédé d'isolement de cellules, procédé de production de cellules souches pluripotentes induites et procédé de production de cellules différenciées
WO2014125955A1 (fr) * 2013-02-15 2014-08-21 国立大学法人京都工芸繊維大学 Procédé pour le repliement d'un anticorps, procédé de production d'un anticorps replié, anticorps replié et leurs utilisations
EP3273241B1 (fr) * 2015-03-17 2019-10-30 National Institute of Advanced Industrial Science and Technology Procédé et kit pour détecter une cellule souche

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009101807A1 (fr) * 2008-02-14 2009-08-20 National University Corporation Kyoto Institute Of Technology Peptide, utilisation du peptide, procédé de fabrication du peptide, support solide ayant le peptide immobilisé sur celui-ci et procédé de fabrication du support solide
JP2011168505A (ja) * 2010-02-16 2011-09-01 Kyoto Institute Of Technology ポリカーボネートおよび／またはポリメタクリル酸メチル親和性ペプチド、およびその利用
WO2013065302A1 (fr) * 2011-11-01 2013-05-10 独立行政法人産業技術総合研究所 Procédé de détection de cellules indifférenciées et procédé de détection d'hydrate de carbone complexe
WO2013122061A1 (fr) * 2012-02-13 2013-08-22 国立大学法人京都工芸繊維大学 Peptide ayant une affinité pour le nitrure de silicium (si3n4) et son utilisation
WO2016129695A1 (fr) * 2015-02-13 2016-08-18 国立大学法人京都工芸繊維大学 Peptides ayant une affinité pour le polydiméthylsiloxane et utilisations associées

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PROPHETER D.C. ET AL.: "Fabrication of an oriented lectin microarray", CHEMBIOCHEM, vol. 11, 7 June 2010 (2010-06-07), pages 1203 - 1207, XP055558760, Retrieved from the Internet <URL:https://doi.org/10.1002/cbic.201000106> *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020218427A1 (fr) * 2019-04-25 2020-10-29 日産化学株式会社 Procédé d'évaluation de la qualité de cellules souches somatiques
WO2021070934A1 (fr) 2019-10-11 2021-04-15 国立研究開発法人産業技術総合研究所 Procédé de dépistage du cancer
JPWO2021070934A1 (fr) * 2019-10-11 2021-04-15
EP4043881A4 (fr) * 2019-10-11 2022-11-23 National Institute Of Advanced Industrial Science and Technology Procédé de dépistage du cancer
JP7656829B2 (ja) 2019-10-11 2025-04-04 国立研究開発法人産業技術総合研究所 がんの検査方法
JP2022122816A (ja) * 2021-02-10 2022-08-23 東ソー株式会社 フコース結合性タンパク質の製造方法
WO2025169902A1 (fr) * 2024-02-05 2025-08-14 国立研究開発法人産業技術総合研究所 Procédé pour détecter ou éliminer les cellules indifférenciées restant dans une population de cellules neurales

Also Published As

Publication number Publication date
JP7228163B2 (ja) 2023-02-24
US20200123222A1 (en) 2020-04-23
JPWO2018190357A1 (ja) 2020-02-27
US11078254B2 (en) 2021-08-03
TW201841935A (zh) 2018-12-01
JP2023011764A (ja) 2023-01-24

Similar Documents

Publication Publication Date Title
JP7228163B2 (ja) レクチンの固定化方法
CN104245961B (zh) 用于微生物检测的方法和系统
JP6083649B2 (ja) 未分化細胞検出方法及び複合糖質検出方法
JP5628793B2 (ja) 生物学的試料中の物質を検出する方法
EP2267151A1 (fr) Procédé de titration de virus
KR101753580B1 (ko) 브루셀라 캐니스 항원의 제조 방법
JP5647599B2 (ja) 生物学的試料中の物質を検出する方法
JPWO2013018836A1 (ja) 生物学的試料中の物質を検出する工程において、非特異的結合を抑制する方法、当該方法に使用するための剤
JP7697024B2 (ja) サンプル検体の検出のための構造体及び方法
JP2013528283A (ja) ヒト睾丸および副睾丸で発現される３０５種類の生殖関連精子局在タンパク質の検出方法
KR101032956B1 (ko) 수청바이러스 유래 뉴클레오캡시드 단백질을 이용하여 신증후출혈열 특이 ＩｇＭ과 ＩｇＧ를 검출하는 신속 진단 키트
US20110039281A1 (en) Method of immunological analysis for detection of antibodies against human gstt1 (anti-hgstt1)
JP7523129B2 (ja) ヘリコバクター・ピロリ菌株の同定方法、および同定用キット
KR101311736B1 (ko) 샌드위치 면역분석 바이오칩 및 이를 사용한 면역분석법
TWI521063B (zh) 生物感測裝置及分離生物分子的方法
Liu et al. Fluorescence-encoded polystyrene microspheres for the application of suspension array technology
WO2016009971A1 (fr) Mesure simplifiée d&#39;un anticorps anti-récepteur de phospholipase a2
US20100255511A1 (en) Immunological tests for the presence of bacteria which make use of antibodies obtained using a specific method
CN110437341A (zh) 一种具有红色荧光活性的检测蛋白及其应用
CN119979546A (zh) 一种检测人血清/脑脊液gfap抗体的方法
CN120112542A (zh) 针对亨廷顿舞蹈症的早期诊断的多肽探针和试剂盒
CN113831401A (zh) 一种sle抗原表位多肽及其在sle诊断中的作用
JPH07301633A (ja) Ｈｉｖ関連抗体測定法
CN106771227A (zh) 生物样本库快速鉴定自身免疫性荨麻疹样本的方法
HK1154408A (en) Virus titration method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18783754

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019512540

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18783754

Country of ref document: EP

Kind code of ref document: A1